TUNEL staining was performed with In situ Cell Detection Kit POD obtained from Roche Diagnostics (Indianapolis, IN) following the manufacturer’s instructions. Endogenous peroxidase blocking with 3% hydrogen peroxide in methanol for 10 minutes was followed by 0.1% triton-X100 in 0.1% sodium citrate for 2 min on ice to increase cell membrane permeability. Heat-induced epitope retrieval with citrate buffer (pH 6.0) for 1 minute at 110°C was followed by blocking with 3% BSA and 20% normal bovine serum in 0.1M Tris-HCl (PH=7.5) for 30 minutes. TUNEL reaction solution was applied for 1 hour at 37°C in a humidity chamber, and then mounted with DAPI mounting solution. Negative controls were performed by using label solution. TUNEL positive cells were counted at 40X magnification. Twenty randomly selected sections covering at least 1,000 cells were counted. Four-five animals were analyzed for each group.
In situ cell detection kit pod
Manufactured by Roche
Sourced in Switzerland, Germany
The In situ Cell Detection Kit POD is a laboratory product designed for the detection of cells in tissue samples. It provides a reliable method for identifying and analyzing cells within their native tissue environment. The kit utilizes a histochemical staining technique to visualize and quantify specific cell types, allowing for further investigation and understanding of cellular processes.
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3 protocols using in situ cell detection kit pod
1
TUNEL Assay for Apoptosis Detection
2
Immunohistochemical Analysis of Cellular Markers
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The immunohistochemical procedure has been described elsewhere (30 (link)). Briefly, slides were incubated with primer antibodies that are mouse monoclonal PCNA (1:1000, Cell Signaling), rabbit monoclonal Ki67 (1:100 dilution, Abcam) and mouse monoclonal human anti-Mitochondrial Antibody (1:250 dilution, Abcam), IL-6 (1:100 dilution, Abcam) and Collagen type I (1:100 dilution, Novus), through overnight at 4°C. Staining was completed by performing LSAB 2 System-HRP (Dako) and then AEC system was used for developing. Hematoxylin counterstaining was performed. Apoptosis was determined by terminal transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay (In situ cell detection kit-POD, Roche, Risch-Rotkreuz, Switzerland) in paraffin-embedded tissue sections according to protocol of the manufacturer.
3
Assessing Follicular Apoptosis via TUNEL
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To determine the development of follicles, TUNEL assays were carried out by a commercial kit (In Situ Cell Detection Kit-POD, Roche, Germany) according to the manufacturer’s instructions. Paraffin-embedded ovarian sections were deparaffinized and hydrated, and then they were processed for blocking endogenous peroxidase activity and antigen retrieval pretreatment. After the slides were blocked with 1% bovine serum albumin for 2 h, they were incubated with the TUNEL reaction mixture in a humid chamber for 1 h at 37 °C. Positive signals were developed with DAB solution (ZLI-9018; ZSGB-BIO, China). Counterstaining was conducted with hematoxylin. Finally, the slides were observed under a light microscope.
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